The research supported by this grant has led to the development of new mass spectrometry techniques for the identification of peptide antigens presented by the MHC molecules, the elucidation of antigen processing pathways that generate them, and the demonstration that many peptides are post-translationally modified. Two areas of particular interest are: 1) the identification of MHC associated peptides that are phosphorylated based on modification of the source protein from which they are derived, and 2) the identification of peptide antigens displayed on melanoma cells in the context of class I MHC molecules, the demonstration that one of these has undergone a post-translational modification, and the description of some aspects of the processing pathway that leads to its presentation. In the present application, we will pursue comprehensive investigations relevant to each of these observations. In specific aim 1, we will initiate a survey of phosphopeptides that are displayed on human cancer cell lines in the context of HLA-A*0201 and HLA-B7. We will identify those peptides that are commonly expressed in cancer versus normal cells and cancers of similar histological type, and evaluate their immunogencity using human class l MHC transgenic mice and human mononuclear cells. We will also analyze phosphopeptide expression in a murine melanoma cell line and identify peptides that may be used for a preclinical evaluation of the efficacy of phosphopeptide based immunotherapies. In specific aim 2, we will evaluate the processing of both class II MHC presented epitopes that are derived from tyrosinase, and that are the targets of T cell responses in melanoma patients. Using mass spectrometry we will identify intermediates in the processing of the Tyr369 epitope, which undergoes deamidation, and determine the relationship of these intermediates to this modification. Based on new understanding of the cell biology of this protein, we will also evaluate how protein folding, interaction with other proteins, and subcellular targeting influence the level of expression of both class I and class epitopes from tyrosinase. Collectively, these studies will continue to use mass spectrometry to offer insight into the display of peptides by MHC molecules of cancer cells, and the relevance of this display to control of tumors by the immune system.